Figure 1.
RNAi screen procedure and control.
(A) A flow chart of the screening process. For the primary screen, lentiviruses expressing shRNAs against 134 genes, packaged in 96-well plates, were used to transduce C2C12 myoblasts seeded in 96-well plates. After 2 days of puromycin selection, the cells were induced to differentiate for 3 days, at the end of which they were fixed and stained for MHC and DAPI. shRNAs that induced morphological changes detectable by visual inspection were subjected to secondary screen with C2C12 cells seeded in 12-well plates, following the procedure described above. Quantification of myotube formation was then performed. (B) As a positive control, Cxcl12 was included in the screen and recovered as a positive hit. Shown are results of the secondary screen with two independent shRNAs. Cells were stained for MHC and DAPI, pseudo-colored green and red, respectively. A non-targeting shRNA served as a negative control. (C) Myotube formation in B was quantified for differentiation index, fusion index and average nuclei number per myotube (see Material and Methods for definition). Data shown are mean ± SD (n = 3). Paired t test was performed to compare data to control. *P<0.05. Scale bar: 100 µm.
Figure 2.
Knockdown of Cxcl9 impairs overall myoblast differentiation.
C2C12 myoblasts were transduced overnight with lentiviruses expressing shRNAs for Cxcl9, selected by puromycin for 2 days, and differentiated for 3 days. (A) At the end of differentiation, the cells were fixed and immuno-stained for MHC (green), and DAPI stain (red) identified nuclei. Scale bar: 100 µm. (B) Myotube formation in A was quantified for differentiation index, fusion index and average nuclei number per myotube. (C) Before differentiation, total RNA was isolated from transduced and selected cells, and subjected to RT-PCR. Data shown are mean ± SD (n = 3). Paired (for B) or one-sample (for C) t tests were performed to compare data to control. *P<0.05. **P<0.01.
Figure 3.
Knockdown of Gdf15 or Scgb3a1 impairs myoblast fusion.
C2C12 myoblasts were transduced overnight with lentiviruses expressing shRNAs for Gdf15 (A-C) or Scgb3a1 (D-E) as described in Fig. 2 legend. (A) MHC (green) and DAPI (red) staining of Gdf15 knockdown cells at the end of 3-day differentiation. (B) Quantification of myotube formation shown in A. (C) RT-PCR results for Gdf15 mRNA. (D) MHC (green) and DAPI (red) staining of Scgb3a1 knockdown cells at the end of 3-day differentiation. (E) Quantification of myotube formation shown in D. Data shown are mean ± SD (n = 3). One sample (C) or paired (B & E) t tests were performed to compare data to control. *P<0.05. **P<0.01. Scale bars: 100 µm.
Figure 4.
Knockdown of Cxcl10 enhances myoblast differentiation.
C2C12 myoblasts were transduced overnight with lentiviruses expressing shRNAs for Cxcl10 as described in Fig. 2 legend. (A) RT-PCR results for Cxcl12 mRNA. (B) MHC (green) and DAPI (red) staining of Cxcl10 knockdown cells at the end of 3-day differentiation. Scale bar: 100 µm. (C) Quantification of myotube formation shown in B. Data shown are mean ± SD (n = 3). One sample (A) or paired (C) t tests were performed to compare data to control. *P<0.05.
Figure 5.
Knockdown of TNFα enhances myoblast differentiation.
C2C12 myoblasts were transduced overnight with lentiviruses expressing shRNAs for TNFα as described in Fig. 2 legend. (A) RT-PCR results for TNFα mRNA. (B) MHC (green) and DAPI (red) staining of TNFα knockdown cells at the end of 3-day differentiation. Scale bar: 100 µm. (C) Quantification of myotube formation shown in B. Data shown are mean ± SD (n = 3). One sample (A) or paired (C) t tests were performed to compare data to control. *P<0.05.
Figure 6.
Knockdown of IL1f9 enhances myoblast differentiation without increasing myotube size.
C2C12 myoblasts were transduced overnight with lentiviruses expressing shRNAs for IL1f9 as described in Fig. 2 legends. (A) RT-PCR results for IL1f9 mRNA. (B) MHC (green) and DAPI (red) staining of IL1f9 knockdown cells at the end of 3-day differentiation. Scale bar: 100 µm. (C) Quantification of myotube formation shown in B. Data shown are mean ± SD (n = 3). One sample (A) or paired (C) t tests were performed to compare data to control. *P<0.05.
Table 1.
RNAi screening identifies potential regulators of myoblast differentiation.
Figure 7.
Validation of expression and knockdown of 6 additional candidate genes.
C2C12 myoblasts were transduced overnight with lentiviruses expressing shRNAs as indicated. After 2-day puromycin selection, total RNA was extracted and subjected to RT-PCR. The results were quantified by densitometry and normalized to β-actin control. One sample t test was performed to compare each data point to control. Data shown are mean ± SD (n = 3). *P<0.05.